Whole blood diluting solution

ABSTRACT

A whole blood diluting solution for analyzing quantitatively whole blood (even when it contains a subject component in an abnormally high quantity, or it is used as a sample after a several-hour hapse from blood-gathering) by supplying a given volume of whole blood sample in the form of a diluted solution to a dry analysis material, which has at least one porous layer side, with the diluting solution containing a water-insoluble dispersed phase (made up of, e.g., macromolecular substances), preferably in an emulsified or suspended condition isotonic to whole blood.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a diluting solution for diluting awhole blood sample, if needed, in analyzing a target component oranalyte in the quantitative analysis contained in the whole bloodsample, using an element for dry analysis. More particularly, it isconcerned with a diluting solution and an analytical process using saiddiluting solution for the quantitative analysis of diluted whole bloodsamples using, for example, analysis element comprising paperimpregnated with a color-producing reagent, or a multilayer analysiselement comprising a light-permeable, water-impermeable support havingat least one reagent layer and an outermost porous layer.

2. Description of the Prior Art

Dry analysis materials or elements and methods for analyzingquantitatively aqueous liquid samples using them are described in U.S.Pat. Nos. 3,552,928, 3,368,872, 3,036,893, 3,016,292 and 2,846,808, andso on.

Multilayer dry analysis elements comprising a transparent support havingthereon at least one reagent layer and a porous layer, in this order,and quantitative analysis methods of aqueous liquid samples using thosematerials are described, e.g., in, Japanese Patent Application (OPI)Nos. 53888/74 137192/75, 140191/76, 3488/77, 131089/78, 101398/79,90859/80, 164356/80, 24576/81, etc. (as used herein "OPI" means an"unexamined published application"), H. G. Curme et al and R. W. Spaydet al., Clinical Chemistry, vol. 24, pp. 1335-1350 (1978), Bert Walter,Anal. Chem., vol. 55, No. 4, pp. 498-514, and so on. These referencesdescribe the possibility of using non-diluted serum, blood plasma anddiluted whole blood as a sample.

More specifically, examples of a clinical test to determine bloodglucose using non-diluted whole blood as a sample and a multilayeranalysis element as the analysis means are described in Ohkubo et al.,Clinical Chemistry, vol. 27, pp. 1287-1290 (1981).

In the clinical application of the whole blood sample analysis using amultilayer dry analysis material, satisfactory analysis results wereobtained when fresh whole blood having normal ranges for the hematocritvalue and fluidity was employed as a sample. However, it was found thatsubstantial lowering of precision and accuracy of the analysis resultsoccurred frequently when whole blood containing a component to beanalyzed in an abnormally high content, having an abnormally highhematocrit value, or having low fluidity was employed as a sample, orwhole blood was used as a sample after a several-hour lapse from bloodsampling. The above problems did not occur for blood serum or bloodplasma, even when the dry analysis method was used. However, rapidityand simplicity of the dry analysis method are lost, since the samplemust be centrifuged to obtain blood serum or blood plasma. Indetermining an analyte in blood, there is much need of obtaining theresult of analysis simply and immediately using the whole blood as itis, as a sample, and a dry analysis element as an analyzing implement.

When whole blood containing a component to be analyzed in an abnormallyhigh concentration, whole blood having an abnormally high hematocritvalue, or whole blood having low fluidity is handled, it is conventionalto dilute the whole blood with an appropriate aqueous diluting solution.When whole blood is diluted, it is desirable that the diluting solutionshould be a solution having, substantially no hemolytic function, andfurther, does not cause agglutination of red corpuscles by dilution sothat no change in fluidity of the whole blood sample occurs. As for theaqueous diluting solutions having the above characteristics, isotonicsolutions containing inorganic salts, such as, physiological saline(including physiological salt solution, Ringer's solution, etc.), andisotonic solutions having a viscosity adjusted properly by addition of ahydrophilic or water-soluble organic substance, such as dextran,polyvinyl pyrrolidone, albumin, etc., to physiological saline havegenerally been employed. However, whole blood samples diluted with theseaqueous diluting solutions exhibit responses different from their neatwhole blood samples with respect to, e.g., color development of ananalyte applied to a dry analysis element. That is, the analyteconcentration calculated by multiplying the concentration derived fromthe calibration curve determined using neat whole blood samples, by thedilution factor, is not exact. The concentration-dilution factorrelationship to correct the above-described error is so complicated thatthe dilution with the isotonic solutions described above is impractical.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a diluting solutionwhich can produce satisfactory results in quantitative analyses using adry analysis element of not only normal whole blood but also abnormalblood, wherein the blood sample is diluted to a definite volume withsaid diluting solution. It is a further object of the present inventionis to extend an applicable scope of dry analysis elements to whole bloodsamples, particularly, to abnormal whole blood samples.

More specifically, the object of the present invention is to achieveanalytical precision and accuracy equivalent to those attainable fornormal whole blood samples, by dilution with a diluting solution of agathered sample, in the case where the sample to be analyzed is

(1) a whole blood sample which contains a subject component of analysisin an amount beyond the upper limit of measurable range,

(2) a whole blood sample having an abnormally high hematocrit value,

(3) a whole blood sample having a low fluidity, or

(4) a whole blood sample which has been allowed to stand for severalhours or longer after blood-sampling.

In accordance with the invention, the above-described objects areattained by using as the whole blood diluting solution, a dilutingsolution containing a water-insoluble dispersed phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and FIG. 2 are schematic diagrams for illustrating a method ofanalyzing a whole blood sample using a multilayer film for dry analysis.

FIG. 3 is a correlation graph obtained by plotting the dGlucorodervalues against the (A×4) values of Table 1. (In FIGS. 3 and 4,triangular marks (Δ) designates data of whole blood samples having ahematocrit value of 20%, square marks (□) designates those having ahematocrit value of 40%, and round marks (O) those having a hematocritvalue of 60%.)

FIG. 4 is a correlation graph obtained by plotting the Glucoroder valueagainst the whole blood (undiluted) value of Table 1.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, a method of whole blood analysis using a multilayeranalysis element is illustrated schematically. The exemplifiedmultilayer analysis element is an analysis film having a multilayerstructure provided with a reagent layer 2, light-reflecting layer 3 anda porous spreading layer 4 on one side of a transparent support 1(wherein, of course, plural reagent layers, a barrier layer, a scavengerlayer, a buffer layer, a detector and so on may be interposed betweenthe support and the spreading layer, if needed). When a whole bloodsample 13 having a concrete or filtrable component 11 and a liquidcomponent 12 in the form of a spot is put on the porous spreading layer4 of a multilayer analysis film, the whole blood spreads rapidly in acircle over the porous spreading layer. The area of the circle isapproximately proportional to the quantity of sample put thereon. Theliquid component then passes through the porous spreading layer as theconcrete component is filtered out, and successively passses through thelight-reflecting layer 3 and arrives at the reagent layer 2. Inprinciple, a selective colorproducing reagent capable of reacting onlywith an analyte in the blood is incorporated in the reagent layer inadvance, so that color development takes place in proportion to thecontent of the analyte. The optical density of the color in thecolor-developed region 14 is measured with a colorimeter from thesupport side, and the content of the analyte in the blood can bedetermined by colorimetry.

The porous spreading layer 4 is made up of a material selected fromamong membrane filter-form nonfibrous isotropic porous materials, porousmaterials made of powdery granules, textiles, paper, and so on. Thespreading layer is capable of a metering or spreading function such thatwhen a drop of aqueous liquid sample is placed thereon, the aqueousliquid spreads rapidly in a circle in the horizontal direction and thenpenetrates in vertical direction, and the aqueous liquid is supplied tothe reagent layer located thereunder in an approximately constant volumeper unit area. In particular, using a textile having a construction madeup of fine granules and containing continuous vacant spaces therein, asdescribed in Japanese Patent Application (OPI) No. 90859/80, and thelike, enables the quantitive analysis of whole blood, because thosematerials possess a spreading function with a respect to not only bloodplasma and serum, but also, whole blood containing a concrete component.

A whole blood diluting solution to be used in the present invention,which contains a water-insoluble dispersed phase, is described in detailbelow.

FIG. 2 illustrates schematically the phenomenon wherein a concretecomponent in the whole blood sample (e.g., red blood corpuscles) isfiltered out by the porous spreading layer to remain on the surface andthe inner part near the surface of the spreading layer, while a liquidcomponent is spreaded by passing through the spreading layer, andfurther passes through the light-reflecting layer, and reaches thereagent layer.

When the water-insoluble dispersed phase is a solid phase, it can bemade up of a substance selected from a group consisting of styrenehomopolymer, copolymers prepared from styrene and monomerscopolymerizable with styrene, acrylate homopolymers, copolymers preparedfrom acrylates and monomers copolymerizable with acrylates, vinylacetate homopolymer, copolymers prepared from vinyl acetate and monomerscopolymerizable with vinyl acetate, vinyl chloride homopolymer,copolymers prepared from vinyl chloride and monomers copolymerizablewith vinyl chloride, red blood corpuscles, and ghosts of red bloodcorpuscles.

When the water-insoluble dispersed phase is a polymer or a copolymer,the molecular weight thereof ranges preferably from 1×10⁴ to 1×10⁶.Preferable monomer copolymerizable with styrene, acrylate, vinylacetate, or vinyl chloride described above is at least one monomerselected from the group consisting of acrylic acid, acrylonitrile,acrylamide, ethylene and maleic acid.

When the water-insoluble dispersed phase is a liquid phase, it can bemade up of a substance selected from a group consisting of adipates,sebacates, trimellitates and phosphates. Details of these substances aredisclosed in Japanese Patent Application (OPI) No. 122956/81.

The alcohol moiety composing the above esters preferably includes astraight or branched alcohol having from 4 to 10 carbon atoms.

The diluting solution contains preferably from 10 to 50 wt %, morepreferably from 15 to 30 wt % of the water-insoluble dispersed phase.

The undiluted whole blood sample may be diluted to preferably from 2 to10 times, more preferably from 3 to 5 times by volume, using thediluting solution.

The above-described water-insoluble dispersed phase is an emulsion or asuspension having a particle size which ranges from 0.01 micron to 10microns. So long as the phase to be dispersed can be mixed homogeneouslywith a water phase by a simple stirring operation, the phase may beemployed in the present invention, Thus, the dispersed phase is notalways required to be a stable dispersion. However, it is more desirablethat the dispersed phase assumes the form of stable suspension oremulsion. Optionally, known additive, such as, surface activesubstances, defoaming agents, antiseptics, etc. can be added to theaqueous diluting solution so long as the addition does not causeinterference with the intended analyses. Organic solvents which can beused in the invention include alcohols, such as, methanol, ethanol,benzyl alcohol, etc. Other organic liquid substances can be also addedto the aqueous diluting solution.

As suitable examples of antiseptics, mention may be made ofparachlorophenol derivatives and benzothiazole derivatives as describedin Japanese Patent Application Nos. 58765/86 and 89348/86.

In addition, a glycolysis inhibitor, agglutination inhibitor and likeadditives are generally added to whole blood taken from a human oranimal body immediately after blood sampling, except when the sampledwhole blood is immediately subjected to analysis.

Dilution can be carried out by (1) adding an aqueous diluting solutionto whole blood, (2) adding whole blood to an aqueous diluting solution,or (3) pouring both whole blood and an aqueous diluting solution almostsimultaneously into a third vessel. After the whole blood and theaqueous diluting solution are combined, the resulting mixture ispreferably submitted to gentle stirring or shaking in order to form ahomogeneous mixture of the aqueous diluting solution with the plasmacomponent in the whole blood, and particularly, the concrete componentin the whole blood. For the purpose of simplifying a diluting operationof whole blood, it is possible, for example, to suck up a desired volumeof aqueous diluting solution with a droplet forming instrument, such as,a micropipette, and subsequently to suck up a definite volume of wholeblood with the same micropipette to produce a mixture of the components.When the dilution is carried out in the described manner, one canreadily proceed to placing the diluted whoe blood in a drop on theporous spreading layer as described hereinafter.

In accordance with the operation techniques described in the above-citedpatent specifications and literatures, a drop of diluted whole bloodsample is then placed on the porous spreading layer of a dry analysismaterial and incubated, if necessary. The optical density of the colordeveloped area is measured by a reflex photometry, and a content of theanalyte in the whole blood sample is determined according to theprinciple of the colorimetric method. Also, it is possible to carry outthe photometry of the color-developed area of the dry analysis materialusing a fluorometric technique. The analyte content in the diluted wholeblood sample is determined first. Since the analyte content in thediluted whole blood sample is determined at once by measuring theoptical density of the color-developed region and using the samecalibration curve obtained from the undiluted whole blood sample,analyte content in the undiluted whole blood sample can be determinedonly by multiplication of the content in the diluted sample by thedilution factor. When the dilution factor is set for 2, 3, 4 or 5, themultiplication becomes very easy.

The hematocrit value of whole blood varies widely with the individualand therefore, a proportion of the volume occupied by blood plasma inwhole blood fluctuates widely depending on the hematocrit value, too.Nevertheless, an analyte content in the undiluted whole blood sample canbe derived from the analyte content in the diluted whole blood sample byusing only the entire volume of the whole blood sample and the dilutionfactor which is a remarkable characteristic of the method of the presentinvention. In addition, application of the whole blood diluting solutionof the present invention is not confined to materials for drycolorimetric analysis. It is also possible to apply it to analysismethods which treat whole blood as a sample and use a chemical sensorutilizing an oxygen electrode, a carbon dioxide electrode, a pHelectrode, an enzyme electrode, a filed effect transistor (FET) or soon.

The present invention is illustrated in more detail by reference to thefollowing example.

EXAMPLE 1

The glucose concentration in whole blood was determined in the followingmanner, in which a diluting solution for diluting whole blood and aquantitative multilayer analysis film for glucose were employed.

Fresh human blood to which heparin was added immediately afterblood-gathering was centrifuged to separate it into a plasma componentand a corpuscle component. These two components were taken out of theapparatus separately, and then mixed in various ratios to repreparewhole blood samples having hematocrit values within the range of 20% to60%. Further, glucose was added and dissolved into each whole bloodsample in an amount necessary to adjust the glucose concentration toabout 100 to 1600 mg/dl. Thus, 30 kinds of whole blood samples as shownin Table 1, which differed in hematocrit value and glucoseconcentration, were prepared.

Separately, a basic solution for an aqueous diluting solution, which hadthe following composition, was prepared.

    ______________________________________    Composition of Basic Solution    for Aqueous Diluting Solution    ______________________________________    NaCl                  9      g    Distilled Water       360    g    Cebian A*             640    g    Suraofu 72N**         0.2    g    ______________________________________     * Products of Daicel Ltd. (Copolymer of styrene and ethylacrylate (1:4     molar ratio), molecular weight: about 1 × 10.sup.5, solid content:     30 wt %, average particle diameter: 0.3 μ)     ** antiseptics produced by Takeda Chemical Industries Ltd.

A diluted whole blood sample was prepared by adding 300 microliter ofthe aqueous diluting solution to 100 microliter of the above-describedwhole blood sample to dilute the whole blood sample exactly 4 times byvolume ratio.

Further, a slide for dry chemical analysis of glucose was made in thefollowing manner.

On a 180 μm-thick smooth film of polyethylene terephthalate having anundercoat of gelatin, a coating solution for forming a reagent layer,which had the following composition, was coated so as to have a drythickness of 15 microns, and dried.

    ______________________________________    Gelatin                 20     g    Peroxidase              2500   IU    Glucose Oxidase         1000   IU    1,7-Dihydroxynaphthalene                            0.5    g    4-Aminoantipyrine       0.5    g    Polyoxyethylene Nonylphenol                            0.2    g    Water                   200    ml    ______________________________________

On the reagent layer, was coated a coating solution for forming a lightshielding layer, which had the following position, so as to have a drythickness of 7 microns, and dried.

    ______________________________________    Gelatin               10     g    Titanium Dioxide      100    g    Water                 500    ml    ______________________________________

On the light shielding layer, an adhesive layer having the followingcomposition was coated so as to have a dry thickness of 2 μm, and dried.

    ______________________________________    Gelatin                 4      g    Polyoxyethylene Nonylphenol                            0.1    g    Water                   200    ml    ______________________________________

The adhesive layer was moistened with water in a quantity of 30 g/m²,and cotton broadcloth was adhered to it by applying light pressurethereon, followed by drying.

The glucose analyzing film made in the above-described manner was cutinto pieces measuring 15 mm ×15 mm in size, and each piece was put on athermoplastic resin flame measuring 24 mm by 28 mm in size.

As for the 30 kinds of whole blood samples and the 30 kinds of dilutedwhole blood samples, a six micro liter portion was collected from eachsample with a micropipette, a droplet of each was placed on thedeveloping layer of the above-described analysis slide, and thenincubated at 37° C. for 6 minutes. Thereafter, the glucose concentrationwas determined using the optical reflection density measured on the PETfilm side with a photometer using a colorimetric method. The dataobtained are set forth in the third and forth columns of Table 1. Thevalues calculated by multiplying the glucose concentrations in thediluted whole blood samples by 4, which are to be used as those ofundiluted samples, are set forth in the extreme right column.

On the other hand, each whole blood sample was centrifuged, and theresulting blood plasma was examined for glucose concentration using aGlucoroder (made by Shino Test Co., Ltd.), or a glucose analyzer basedon an enzyme electrode process. The results obtained are set forth atthe second column from the left. The term Hct in the table refers to ahematocrit value.

                  TABLE 1    ______________________________________    Gluco-    Whole Blood    roder     Sample     Whole Blood Sample    Data      (undiluted)                         diluted 4 times (A)                                        A × 4    ______________________________________    Hct 20     1     63      69         20           80     2    165     164         48          192     3    263     222         62          248     4    441     421        111          444     5    582     600        151          604     6    764     impossible 191          764                  to measure     7    924     "          240          960     8    1146    "          290          1160     9    1290    "          313          1252    10    1422    "          334          1336    Hct 40    11    120     130         27          108    12    222     222         56          224    13    327     322         91          364    14    531     506        128          512    15    735     540        180          720    16    933     548        239          956    17    1149    544        302          1208    18    1308    513        323          1292    19    1530    525        367          1468    20    1623    489        397          1588    Hct 60    21     75      75         23           92    22    192     190         50          200    23    264     259         70          280    24    480     344        120          480    25    684     363        171          684    26    903     350        222          888    27    1002    363        236          944    28    1347    347        321          1284    29    1400    375        341          1364    30    1647    373        403          1612    ______________________________________

As can be seen from the data in Table 1, and as depicted in FIG. 4, theconventional method in which glucose concentration is determined throughputting a drop of undiluted whole blood sample on a multilayer materialfor dry analysis has a tendency to produce glucose concentration valueslower than those obtained by a Glucoroder based on an enzyme electrodeprocess. This latter method has been prevailingly used for measurementof glucose concentration in blood when whole blood samples having highglucose concentrations are analyzed. This tendency becomes morepronounced as the hematocrit value becomes higher. In contrast to theconventional method, the glucose concentration value determined with themethod of the present invention in which a whole blood sample, diluted 4times with the whole blood diluting solution of the present invention,is put in droplet form on a multilayer material for dry analysisprovided data, agree well with those obtained by the enzyme electrodeprocess even in the high glucose concentration region and further in thehigh hematocrit value region, as is apparent from FIG. 3.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A whole blood diluting solution for use inanalyzing whole blood quantitatively by applying a given volume of awhole blood sample in the form of diluted solution to an element for dryanalysis having at least one porous layer and containing at least onereagent onto the porous layer side, said diluting solution containing adispersed phase insoluble in water.
 2. The whole blood sample dilutingsolution of claim 1 wherein the particle size of said dispersed phaseinsoluble in water ranges from 0.01 micron to 10 microns.
 3. The wholeblood sample diluting solution of claim 1 wherein said dispersed phaseinsoluble in water is contained in a proportion of from 10 to 50%. 4.The whole blood sample diluting solution of claim 1, wherein saiddispersed phase insoluble in water is made up of a macromolecularsubstance selected form the group consisting of styrene homopolymer,copolymers prepared from styrene and monomers capable of copolymerizingwith styrene, acrylate homopolymers, copolymers prepared from acrylatesand monomers capable of copolymerizing with acrylates, vinyl acetatehomopolymers, copolymers prepared from vinyl acetate and monomerscapable of copolymerizing with vinyl acetate, vinyl chloridehomopolymers, copolymers prepared from vinyl chloride and monomerscapable of copolymerizing with vinyl chloride, red blood corpuscles, andghosts of red blood corpuscles.
 5. The whole blood sample dilutingsolution of claim 1 wherein said water-insoluble, dispersed phase is asuspension or an emulsion.
 6. The whole blood sample diluting solutionof claim 1 which is substantially isotonic to whole blood.
 7. In amethod of analyzing quantitatively a particular analyte in a whole bloodsample by the use of a dry analysis element having at least one reagentlayer and a porous layer in fluid contact with the reagent layer whereinthe sample containing the analyte is placed onto the porous layer, theimprovement which comprises diluting the whole blood sample with adiluting solution containing a dispersed phase insoluble in water. 8.The method of claim 7 wherein said analyte is glucose.
 9. The method ofclaim 7 wherein the particle size of said dispersed phase insoluble inwater ranges from 0.01 micron to 10 microns.
 10. The method of claim 7wherein said dispersed phase insoluble in water is contained in aproportion of from 10 to 50%.
 11. The method of claim 7 wherein saiddispersed phase insoluble in water is made up of a macromolecularsubstance selected from the group consisting of styrene homopolymer,copolymers prepared from styrene and monomers capable of copolymerizingwith styrene, acrylate homopolymers, copolymers prepared from acrylatesand monomers capable of copolymerizing with acrylates, vinyl acetatehomopolymers, copolymers prepared from vinyl acetate and monomerscapable of copolymerizing with vinyl acetate, vinyl chloridehomopolymers, copolymers prepared from vinyl chloride and monomerscapable of copolymerizing with vinyl chloride, red blood corpuscles, andghosts of red blood corpuscles.
 12. The method of claim 7 wherein saidwater-insoluble, dispersed phase is a suspension or an emulsion.
 13. Themethod of claim 7 wherein the diluting solution is substantiallyisotonic to whole blood.